Domain conformations of the motor subunit of EcoR124I involved in ATPase activity and dsDNA translocation

Bacterial type I restriction-modification systems are composed of three different subunits: one HsdS subunit is required for identification of target sequence and anchoring the enzyme complex on DNA; two HsdM subunits in the methyl-transferase complex serve for host genome modification accomplishing a protective function against self-degradation; two HsdR (or motor) subunits house ATP-dependent translocation and consequent cleavage of double stranded DNA activities. The crystal structure of the 120 kDa HsdR subunit of the Type I restriction-modification system EcoR124I in complex with ATP was recently reported. HsdR is organized into four approximately globular structural domains in a nearly square-planar arrangement: the N-terminal endonuclease domain, the RecA-like helicase domains 1 and 2 and the C-terminal helical domain. The near-planar arrangement of globular domains creates prominent grooves between each domain pair. The two helicase-like domains form a canonical helicase cleft in which double-stranded B-form DNA can be accommodated without steric clash. The helical domain, probably involved in complex assembly, exhibits only a few specific interactions with helicase 2 domain. Molecular mechanism of dsDNA translocation, cleavage and ATP hydrolysis has not been yet structurally investigated. Here we propose a translocation cycle of the restriction-modification system EcoR124I based on analysis of available crystal structures of superfamily 2 helicases, strutural modeling and complementary biochemical characterization of mutations introduced in sites potentially inportant for translocation in the HsdR motor subunit. Also a role of the extended region of the helicase motif III in ATPase activity of EcoR124I was probed

The Effect of Variable Light Source and Light Intensity on the Growth of Three Algal Species

Light is the essential energy source for autotrophically growing organisms, including microalgae. Both light intensity and light quality affect cell growth and biomass composition. Here we used three green algae—Chlamydomonas reinhardtii, Desmodesmus quadricauda, and Parachlorella kessleri—to study the effects of different light intensities and light spectra on their growth. Cultures were grown at three different light intensities (100, 250, and 500 µmol m−2 s−1) and three different light sources: fluorescent lamps, RGB LEDs, and white LEDs. Cultures of Desmodesmus quadricauda and Parachlorella kessleri were saturated at 250 µmol m−2 s−1, and further increasing the light intensity did not improve their growth. Chlamydomonas reinhardtii cultures did not reach saturation under the conditions used. All species usually divide into more than two daughter cells by a mechanism called multiple fission. Increasing light intensity resulted in an increase in maximum cell size and division into more daughter cells. In Parachlorella kessleri cells, the concentration of photosynthetic pigments decreased with light intensity. Different light sources had no effect on algal growth or photosynthetic pigments. The results show a species-specific response of algae to light intensity and support the use of any white light source for their cultivation without negative effects on growth

Diclofenac Alters the Cell Cycle Progression of the Green Alga Chlamydomonas reinhardtii

The aim of the study was to verify the hypothesis that a potential cause of the phytotoxicity of diclofenac (DCF, a non-steroidal anti-inflammatory drug) is an effect of cell cycle progression. This research was conducted using synchronous cultures of a model organism, green alga Chlamydomonas reinhardtii. The project examined DCF effects on selected parameters that characterize cell cycle progression, such as cell size, attainment of commitment points, DNA replication, number of nuclei formed during cells division and morphology of cells in consecutive stages of the cell cycle, together with the physiological and biochemical parameters of algae cells at different stages. We demonstrated that individual cell growth remained unaffected, whereas cell division was delayed in the DCF-treated groups grown in continuous light conditions, and the number of daughter cells from a single cell decreased. Thus, the cell cycle progression is a target affected by DCF, which has a similar anti-proliferative effect on mammalian cells

Supra-Optimal Temperature: An Efficient Approach for Overaccumulation of Starch in the Green Alga Parachlorella kessleri

Green algae are fast-growing microorganisms that are considered promising for the production of starch and neutral lipids, and the chlorococcal green alga Parachlorella kessleri is a favorable model, as it can produce both starch and neutral lipids. P. kessleri commonly divides into more than two daughter cells by a specific mechanism—multiple fission. Here, we used synchronized cultures of the alga to study the effects of supra-optimal temperature. Synchronized cultures were grown at optimal (30 °C) and supra-optimal (40 °C) temperatures and incident light intensities of 110 and 500 μmol photons m−2 s−1. The time course of cell reproduction (DNA replication, cellular division), growth (total RNA, protein, cell dry matter, cell size), and synthesis of energy reserves (net starch, neutral lipid) was studied. At 40 °C, cell reproduction was arrested, but growth and accumulation of energy reserves continued; this led to the production of giant cells enriched in protein, starch, and neutral lipids. Furthermore, we examined whether the increased temperature could alleviate the effects of deuterated water on Parachlorella kessleri growth and division; results show that supra-optimal temperature can be used in algal biotechnology for the production of protein, (deuterated) starch, and neutral lipids

The helical domain of the EcoR124I motor subunit participates in ATPase activity and dsDNA translocation

Type I restriction-modification enzymes are multisubunit, multifunctional molecular machines that recognize specific DNA target sequences, and their multisubunit organization underlies their multifunctionality. EcoR124I is the archetype of Type I restriction-modification family IC and is composed of three subunit types: HsdS, HsdM, and HsdR. DNA cleavage and ATP-dependent DNA translocation activities are housed in the distinct domains of the endonuclease/motor subunit HsdR. Because the multiple functions are integrated in this large subunit of 1,038 residues, a large number of interdomain contacts might be expected. The crystal structure of EcoR124I HsdR reveals a surprisingly sparse number of contacts between helicase domain 2 and the C-terminal helical domain that is thought to be involved in assembly with HsdM. Only two potential hydrogen-bonding contacts are found in a very small contact region. In the present work, the relevance of these two potential hydrogen-bonding interactions for the multiple activities of EcoR124I is evaluated by analysing mutant enzymes using in vivo and in vitro experiments. Molecular dynamics simulations are employed to provide structural interpretation of the functional data. The results indicate that the helical C-terminal domain is involved in the DNA translocation, cleavage, and ATPase activities of HsdR, and a role in controlling those activities is suggested

Functional coupling of duplex translocation to DNA cleavage in a type I restriction enzyme

Type I restriction-modification enzymes are multifunctional heteromeric complexes with DNA cleavage and ATP-dependent DNA translocation activities located on motor subunit HsdR. Functional coupling of DNA cleavage and translocation is a hallmark of the Type I restriction systems that is consistent with their proposed role in horizontal gene transfer. DNA cleavage occurs at nonspecific sites distant from the cognate recognition sequence, apparently triggered by stalled translocation. The X-ray crystal structure of the complete HsdR subunit from E. coli plasmid R124 suggested that the triggering mechanism involves interdomain contacts mediated by ATP. In the present work, in vivo and in vitro activity assays and crystal structures of three mutants of EcoR124I HsdR designed to probe this mechanism are reported. The results indicate that interdomain engagement via ATP is indeed responsible for signal transmission between the endonuclease and helicase domains of the motor subunit. A previously identified sequence motif that is shared by the RecB nucleases and some Type I endonucleases is implicated in signaling

Cleavage of circular DNA.

<p>Circular plasmid DNA bearing one EcoR124I recognition site was reacted with enzymes reconstituted from HsdS₁HsdM₂ methylase and WT HsdR or mutant HsdRs Lys220Glu, Lys220Ala, or Lys220Arg, and analyzed as in Fig 2. OC, open circular product (▲); L, linear product (●); SC, supercoiled substrate (■); C, control plasmid linearized with HindIII. A: Reactions stopped at the indicated time points were applied to 1.2% agarose gels and visualized by ethidium bromide staining. M is the marker of the indicated numbers of basepairs and C is the linearized plasmind DNA as a control B: Quantification. The three indicated DNA species were quantified individually. Plots for the increase of linear DNA product were derived by fitting an exponential rise to maximum function in SigmaPlot. The points are given for quantification of the gels shown in A, and standard deviations are given from the mean of seven repetitions (WT, Lys220Ala, Lys220Glu) or six repetitions (Lys220Arg) of the experiment conducted with independently purified enzyme preparations.</p

ATP contacts.

<p>Models and electron density are shown for A, WT HsdR; B, Lys220Glu chain A; C, Lys220Arg; D, Lys220Ala. Domain segments (ribbons) and selected residues (stick models) are color-coded as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128700#pone.0128700.g001" target="_blank">Fig 1</a>, with Mg ion shown as a green sphere. Electron density (blue mesh) is shown for ATP (upper center of each panel, atomic colors and orange carbon) and for the 220s loop (lower). The electron density for WT HsdR is better at the same contour level due to its higher resolution, with corresponding differences in the electron density mesh spacing. Dashed lines indicate distances short enough to permit bonding interactions between the indicated functional groups.</p

Effect of changes at HsdR Lys220 on the restriction phenotype of EcoR124I.

<p>^a Restriction activity was determined as the efficiency of plating of λvir.0 on the test strains relative to the efficiency of plating of λvir.0 on <i>E</i>. <i>coli</i> JM109(DE3) indicator (nonrestricting) strain as described in Methods. The values are the mean of at least three independent experiments. ^SD The standard deviation</p><p>^b Positive complementation was tested in r− host <i>E</i>. <i>coli</i> JM109(DE3)[pACMS] (r−m+).</p><p>^c Negative complementation was tested in r+ host <i>E</i>. <i>coli</i> JM109(DE3)[pKF650] (r+m+).</p><p>Effect of changes at HsdR Lys220 on the restriction phenotype of EcoR124I.</p

Crystallographic data collection and refinement statistics.

<p>* Values in parentheses are for the highest-resolution shell.</p><p>Crystallographic data collection and refinement statistics.</p